![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
McArdle Laboratory for Cancer Research, Department of Oncology, School of Medicine and Public Health, University of Wisconsin Madison, Madison, Wisconsin
Received for publication March 7, 2008.
Accepted for publication July 31, 2008.
 |
Abstract |
|---|
 Top Abstract Materials and MethodsResultsDiscussionReferences |
|---|
; Eisen et al., 1983). Once ligands such as these bind the receptor, the complex translocates to the nucleus in which the AHR dissociates and dimerizes with its transcriptional partner, the aryl hydrocarbon receptor nuclear translocator (ARNT). In the nucleus, the AHR/ARNT dimer binds to cognate "dioxin-responsive elements" in the enhancer regions of responsive genes, resulting in the transcriptional up-regulation of those genes (Reyes et al., 1992; Henry and Gasiewicz, 1993; Hord and Perdew, 1994). The effects mediated by this pathway include the adaptive up-regulation of certain cytochromes P450, as well as toxic endpoints that include thymic involution, hepatocellular damage, and cleft palate (Poland and Knutson, 1982).
It has been shown recently that the AHR also plays an important role in hepatovascular development (Lahvis et al., 2005). In the developing embryo, the ductus venosus (DV) serves as a shunt that connects the umbilical cord blood with blood from the portal vein and the inferior vena cava (IVC) (Schermerhorn et al., 1996). The DV normally closes within a few hours or days of parturition. Although Ahr(-/-) mice are viable and fertile, a consistent phenotype observed in these animals is the presence of an open, or patent DV throughout life (Lahvis et al., 2005). Failure of DV closure in Ahr(-/-) mouse models causes a decrease in portal blood supply to the liver, thereby limiting nutrients and decreasing overall liver size (Lahvis et al., 2005).
The cytosolic form of the receptor has been shown to be complexed with a dimer of heat shock protein of 90 kDa and either the cochaperone p23 or the aryl hydrocarbon receptor-associated protein-9 (ARA9) (Carver and Bradfield, 1997; Shetty et al., 2003; Hollingshead et al., 2004). ARA9 is also known as the aryl hydrocarbon receptor-interacting protein or the hepatitis B virus X-associated protein 2 (XAP2) (Ma and Whitlock, 1997; Meyer and Perdew, 1999). In an effort to understand whether ARA9 plays a role in the developmental aspect of AHR signal transduction, we used gene targeting techniques to generate a series of hypomorphic alleles at the Ara9 locus in mice. Hypomorphic alleles at Ara9 were used because mice harboring an Ara9-null allele [i.e., Ara9(-/-)] display 100% embryonic lethality as a result of several embryonic heart defects (Lin et al., 2007). Given that Ahr(-/-) mice do not display similar congenital heart defects, we conclude that ARA9 plays a role in heart development that is independent of its role in AHR function (Fernandez-Salguero et al., 1996; Schmidt et al., 1996).
Our use of Ara9 hypomorphic models was based upon two predictions. First, we hypothesized that mice hypomorphic for Ara9 could be developed that would express enough protein to alleviate the developmental block caused by the essential role of the protein in early cardiac development. Second, if ARA9 was essential for AHR developmental signaling, then mice with a lower level of ARA9 protein expression would also display a phenocopy of the Ahr-null animal (i.e., a patent DV). We show here that these criteria can indeed be met. Furthermore, we provide the first evidence that ARA9 is essential for AHR-mediated developmental signaling.
|
|
Materials and Methods |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
CAra9fxneo and is described in Fig. 1 and in previous work from our laboratory (Lin et al., 2007). A BamHI restriction enzyme analysis resulting in fragment sizes of 553, 844, and 11,165 bp was performed to confirm the orientation of components of the targeting vector (PL 2008), including the tetratricopeptide repeat domains, Lox P sites, and arms of homology. Embryonic stem cell culture conditions and genotyping were performed using the same methods as reported previously (Lin et al., 2007).
Visualization of the Ductus Venosus. Mice harboring a patent DV were identified by perfusion of the liver with a solution of trypan blue using methods described previously (Walisser et al., 2004a). The status of the DV was confirmed on a subset of livers via time-lapse angiography using a method described previously (Walisser et al., 2004b).
Western Blot Analysis. Western blots were performed using the same methods described previously (Lin et al., 2007).
Statistics. In experimental analysis of multiple comparisons, an analysis of variance was performed, and Tukey's test was used to determine differences, with a p 
0.05. Statistical analysis of genotype distribution was compared by 
2 analysis (Devore and Peck, 1986).
|
Results |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
2 analysis was not significantly different from expected with a viable allele (p 1; 2 = 0.69) and indicated adherence to the expected Mendelian ratio of 1:2:1. Gross anatomy and histological examinations suggested that wild-type, heterozygote, and homozygous Ara9fxneo animals are outwardly normal (data not shown).
The Ara9fxneo Allele Is Hypomorphic. The expression of ARA9 was measured by Western blot analysis of protein extracts from heart, liver, kidney, spleen, and thymus from wild-type [Ara9(+/+)] and Ara9(fxneo/fxneo) mice (Fig. 2A). Hypomorphs displayed marked decreases in ARA9 protein expression in all tissues examined. Antibodies raised against either the FK506-binding protein or tetratricopeptide repeat domains (Lin et al., 2007) yielded similar results (data not shown). Based on the ARA9 Western blot gradient, expression of the ARA9 protein from the Ara9fxneo allele was estimated to be approximately 10% of the expression of the wild-type allele (Fig. 2B).
|
; Walisser et al., 2004a). In the progeny from appropriate crosses of heterozygous animals, the angiograms of wild-type littermates revealed that the contrast agent flowed through the portal vein and into the branches of the liver and then into the suprahepatic then infrahepatic IVC (Fig. 3A). In contrast, livers of Ahr(-/-) and Ara9(fxneo/fxneo) mice display portocaval shunting between the portal vein and IVC, indicating the presence of a patent DV (Lahvis et al., 2000). Perfusion assays using trypan blue were performed on a larger number of animals and revealed that Ara9(fxneo/fxneo) mice display a patent DV at a frequency of 83%. Mice that are Ara9(+/-) display a DV frequency of 56%. Mice that are Ara9(+/fxneo) display a patent DV with a frequency of approximately 10% (Fig. 3B). Wild-type animals [Ara9(+/+)] do not display a patent DV. No association was noted between the sex of the animal and persistence of the DV (data not shown). Estimates of DV patency in Ara9(-/-) mice could not be obtained because these mice are resorbed or stillborn because of cardiac defects that begin around day E12 (Lin et al., 2007).
|
The Presence of DV Causes a Significant Decrease in Liver Weight. In addition to and as a result of the patent DV, another common phenotype observed in Ahr(-/-) mice is a decreased relative liver weight. To investigate whether this relationship also holds true for the Ara9 hypomorphic lines, we compared relative liver weights from wild-type, heterozygous hypomorphic, homozygous hypomorphic, and heterozygous null animals. As expected, liver weight in Ara9(fxneo/fxneo) and Ara9(+/-) mice were significantly less than in wild-type and Ara9(+/fxneo) mice (Fig. 3B).
|
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
). Based on this observation, it follows that proteins that influence AHR folding, localization, or function may also be essential for AHR-mediated hepatovascular development. The simplest way to test this idea is to create mouse models with null alleles at candidate genes and to examine their AHR-mediated response profile. Unfortunately, most, if not all of the proteins known to be involved in AHR signaling are not amenable to classic gene targeting approaches that use null alleles, because many AHR-associated gene products are essential for more than one independent cellular pathway required for the survival of the animal. A relevant example is the ARNT protein, which is also required for developmental angiogenesis mediated by hypoxia-inducible factors (Maltepe et al., 1997; Chan et al., 1999). For ARNT, we were able to overcome its essential nature and provide evidence for its role in AHR-mediated development by using mice that exhibited decreased expression of ARNT (i.e., hypomorphic expression) (Walisser et al., 2004b). In that model, we found that decreased expression of the ARNT protein allowed for normal embryonic angiogenesis, yet yielded a developmental phenotype that was identical to that observed in the Ahr(-/-) model (i.e., a patent DV).
Given our recent study demonstrating that Ara9(-/-) mice die in fetal development as a result of double-outlet right ventricle and ventricular septal defect, we chose to use a "hypomorph strategy" similar to that used for ARNT (Walisser et al., 2004b; Lin et al., 2007). To this end, we constructed a series of recombinant alleles at the Ara9 locus in mice. Our strategy was based on work from this laboratory and others, which has shown that targeted insertion of the Neo gene into a locus of interest frequently results in the generation of a hypomorphic allele (Walisser et al., 2004b). Given that Neo insertion was an intermediary step in generating a conditional Ara9 allele, a hypomorphic allele of Ara9 was constructed and used to characterize the influence of hypomorphic ARA9 expression on AHR-mediated liver development. It was our prediction the generation of a hypomorphic mouse would allow us to bypass embryonic lethality and enable us to study the effect of decreased ARA9 protein expression on AHR-mediated biology.
Using the Ara9fxneo allele along with our previously generated Ara9 allele, we created an allelic series where graded expression of the ARA9 protein could be achieved after appropriate genetic crosses. The expression levels of ARA9 protein ranged from highest to lowest in Ara9(+/+), Ara9(+/fxneo), Ara9(+/-), and Ara9(fxneo/fxneo) mice, respectively (Fig. 2A; data not shown). Although our Western blot-derived estimates of ARA9 protein expression from these models provides only a semiquantitative measure of relative protein expression, the data do support an estimate of approximately a 10% expression of ARA9 in the Ara9(fxneo/fxneo) mice, a 50% expression in the Ara9(+/-)mice, and a 60% expression in Ara9(+/fxneo) mice relative to wild-type mice (Fig. 2A).
Examination of a large cohort of Ara9 mutants supports our two initial predictions. First, we found that mice homozygous for a hypomorphic Ara9 allele (Ara9(fxneo/fxneo) proceeded through development in normal numbers (Fig. 1), suggesting that 10% of normal expression of ARA9 is sufficient to overcome the block in heart development observed previously in null animals (Lin et al., 2007). Second, we hypothesized that a marked reduction in ARA9 expression would lead to insufficient AHR signaling and that the corresponding mice would display many of the phenotypes of Ahr-null animals. Examination of the developmental phenotype, patent DV, was of particular interest to us. To this point, we measured the frequency of the DV and found 83% of the hypomorphic animals to display this developmental phenotype (Fig. 3). In addition to the phenocopy exhibited by Ara9 hypomorphs and Ahr-null animals, additional support for the importance of ARA9 in AHR-mediated DV closure came from the observation of a "dose effect" of ARA9 expression on these endpoints. A slight reduction of ARA9 protein levels by one copy of Ara9fxneo (i.e., the Ara9(+/fxneo) model) led to only a 10% frequency of patent DV. Intermediate expression of ARA9 from the Ara9(+/-) model display a patent DV incidence of 56%. Even greater reduction of ARA9 protein from two copies of the hypomorphic allele (i.e., Ara9(fxneo/fxneo) led to an 83% incidence of patent DV. In our examination of hundreds of wild-type mice (Ara9), we have never observed a patent DV. In keeping with this, we have observed a 0% frequency of patent DV in our wild-type cohort. Together, these data support the idea that ARA9 plays an important role in AHR-mediated developmental signaling.
Because of the persistence of the DV in ARA9 hypomorphic animals, this model has less power as a tool to study the adaptive and toxic pathways mediated by the AHR. This concept is based upon the observation that the portocaval shunting, which accompanies a patent DV, can lead to aberrant disposition of TCDD and related AHR agonists in many tissues, making a direct comparison between mutant and wild-type responses problematic (Thomae et al., 2004). For example, if we were trying to show a relationship between Ara9 expression and TCDD toxicity for a given liver endpoint, we would predict a decreased potency in the Ara9 hypomorphs. However, if this did occur, we would not know whether the attenuation was due to decreased cellular AHR signaling or to a decreased concentration of TCDD in the hepatic parenchyma as a result of shunting. Therefore, to understand the role ARA9 plays in toxic and adaptive signaling, our future plans include crossing the Ara9fxneo hypomorph with the FLPeR line (mice containing Flp-recombinase driven by the GTSRosa promoter), thereby allowing for germline excision of the neomycin cassette and creation of a conditional ARA9 mouse (Dymecki, 1996). These mice can then be crossed to mice where expression of Cre is driven by the Albumin promoter, allowing for specific excision of the Ara9 allele in hepatocytes (Kellendonk et al., 2000). Such a model will allow us to understand the relationship between hepatic expression of ARA9 and AHR-mediated hepatotoxicity and adaptive metabolism.
|
Acknowledgements |
|---|
|
Footnotes |
|---|
ABBREVIATIONS: AHR, aryl hydrocarbon receptor; ARNT, aryl hydrocarbon receptor nuclear translocator; IVC, inferior vena cava; ARA9, aryl hydrocarbon receptor-associated protein-9; bp, base pair(s); kb, kilobase(s); TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin.
Address correspondence to: Dr. Christopher Bradfield, 1400 University Ave., Madison, WI 53706. E-mail: bradfield{at}oncology.wisc.edu
|
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Chan WK, Yao G, Gu YZ, and Bradfield CA (1999) Cross-talk between the aryl hydrocarbon receptor and hypoxia inducible factor signaling pathways. Demonstration of competition and compensation. J Biol Chem 274: 12115-12123.
Devore J and Peck R (1986) Statistics: The Exploration and Analysis of Data. West Publishing Co., New York.
Dymecki SM (1996) Flp recombinase promotes site-specific DNA recombination in embryonic stem cells and transgenic mice. Proc Natl Acad Sci U S A 93: 6191-6196.
Eisen HJ, Hannah RR, Legraverend C, Okey AB, and Nebert DW (1983) The Ah-receptor: controlling factor in the induction of drug-metabolizing enzymes by certain chemical carcinogens and other environmental pollutants, in Biochemical Actions of Hormones (Litwack G ed), Vol 10, pp 227-257, Academic Press, New York.
Fernandez-Salguero PM, Hilbert DM, Rudikoff S, Ward JM, and Gonzalez FJ (1996) Aryl-hydrocarbon receptor-deficient mice are resistant to 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced toxicity. Toxicol Appl Pharmacol 140: 173-179.[CrossRef][Medline]
Henry EC and Gasiewicz TA (1993) Transformation of the aryl hydrocarbon receptor to a DNA-binding form is accompanied by release of the 90-kDa heat-shock protein and increased affinity for 2,3,7,8-tetracholorodibenzo-p-dioxin. Biochem J 294: 95-101.[Medline]
Hollingshead BD, Petrulis JR, and Perdew GH (2004) The Ah receptor transcriptional regulator XAP2 antagonizes p23 binding to Ah receptor/Hsp90 complexes and is dispensable for receptor function. J Biol Chem 279: 45652-45661
Hord NG and Perdew GH (1994) Physicochemical and immunocytochemical analysis of the aryl hydrocarbon receptor nuclear translocator: characterization of two monoclonal antibodies to the aryl hydrocarbon receptor nuclear translocator. Mol Pharmacol 46: 618-626.[Abstract]
Kellendonk C, Opherk C, Anlag K, Schütz G, and Tronche F (2000) Hepatocyte-specific expression of Cre recombinase. Genesis 26: 151-153.[CrossRef][Medline]
Lahvis GP, Lindell SL, Thomas RS, McCuskey RS, Murphy C, Glover E, Bentz M, Southard J, and Bradfield CA (2000) Portosystemic shunts and persistent fetal vascular structures in Ah-receptor deficient mice. Proc Natl Acad Sci U S A 97: 10442-10447.
Lahvis GP, Pyzalski RW, Glover E, Pitot HC, McElwee MK, and Bradfield CA (2005) The aryl hydrocarbon receptor is required for developmental closure of the ductus venosus in the neonatal mouse. Mol Pharmacol 67: 714-720.
Lin BC, Sullivan R, Lee Y, Moran S, Glover E, and Bradfield CA (2007) Deletion of the aryl hydrocarbon receptor-associated protein 9 leads to cardiac malformation and embryonic lethality. J Biol Chem 282: 35924-35932.
Ma Q and Whitlock JP Jr (1997) A novel cytoplasmic protein that interacts with the Ah receptor, contains tetratricopeptide repeat motifs, and augments the transcriptional response to 2,3,7,8-tetrachlorodibenzo-p-dioxin. JBiolChem 272: 8878-8884.
Maltepe E, Schmidt JV, Baunoch D, Bradfield CA, and Simon MC (1997) Abnormal angiogenesis and responses to glucose and oxygen deprivation in mice lacking the protein ARNT. Nature 386: 403-407.[CrossRef][Medline]
Meyer BK and Perdew GH (1999) Characterization of the AhR-hsp90-XAP2 core complex and the role of the immunophilin-related protein XAP2 in AhR stabilization. Biochemistry 38: 8907-8917.[CrossRef][Medline]
Poland A (1982) Induction of the drug-metabolizing enzymes, in Host Factors in Human Carcinogenesis (Armstrong B and Bartsch H eds) no. 39, pp 351-364. IARC Scientific Publications, Lyon, France.
Poland A and Knutson JC (1982) 2,3,7,8-Tetrachlorodibenzo-p-dioxin and related halogenated aromatic hydrocarbons: examination of the mechanism of toxicity. Annu Rev Pharmacol Toxicol 22: 517-554.[CrossRef][Medline]
Reyes H, Reisz-Porszasz S, and Hankinson O (1992) Identification of the Ah receptor nuclear translocator protein (Arnt) as a component of the DNA binding form of the Ah receptor. Science 256: 1193-1195.
Schermerhorn T, Center SA, Dykes NL, Rowland PH, Yeager AE, Erb HN, Oberhansley K, and Bonda M (1996) Characterization of hepatoportal microvascular dysplasia in a kindred of Cairn terriers. J Vet Intern Med 10: 219-230.[Medline]
Schmidt JV, Su GH, Reddy JK, Simon MC, and Bradfield CA (1996) Characterization of a murine Ahr null allele: involvement of the Ah receptor in hepatic growth and development. Proc Natl Acad Sci U S A 93: 6731-6736.
Shetty PV, Bhagwat BY, and Chan WK (2003) P23 enhances the formation of the aryl hydrocarbon receptor-DNA complex. Biochem Pharmacol 65: 941-948.[CrossRef][Medline]
Thomae TL, Glover E, and Bradfield CA (2004) A maternal Ahr null genotype sensitizes embryos to chemical teratogenesis. J Biol Chem 279: 30189-30194.
Walisser JA, Bunger MK, Glover E, and Bradfield CA (2004a) Gestational exposure of Ahr and Arnt hypomorphs to dioxin rescues vascular development. Proc Natl Acad Sci U S A 101: 16677-16682.
Walisser JA, Bunger MK, Glover E, Harstad EB, and Bradfield CA (2004b) Patent ductus venosus and dioxin resistance in mice harboring a hypomorphic Arnt allele. J Biol Chem 279: 16326-16331.
This article has been cited by other articles:
|
|
 |
|
  P. C. Boutros, K. A. Bielefeld, R. Pohjanvirta, and P. A. Harper Dioxin-Dependent and Dioxin-Independent Gene Batteries: Comparison of Liver and Kidney in AHR-Null Mice Toxicol. Sci., November 1, 2009; 112(1): 245 - 256. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. J. Jenny, S. I. Karchner, D. G. Franks, B. R. Woodin, J. J. Stegeman, and M. E. Hahn Distinct Roles of Two Zebrafish AHR Repressors (AHRRa and AHRRb) in Embryonic Development and Regulating the Response to 2,3,7,8-Tetrachlorodibenzo-p-dioxin Toxicol. Sci., August 1, 2009; 110(2): 426 - 441. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Nukaya, S. Moran, and C. A. Bradfield The role of the dioxin-responsive element cluster between the Cyp1a1 and Cyp1a2 loci in aryl hydrocarbon receptor biology PNAS, March 24, 2009; 106(12): 4923 - 4928. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|
 |
 |
 |
